This invention relates to liquid chromatography and more particularly to an automated system for performing isocratic interactive high performance liquid chromatographic separations and finding the optimum mobile phase strength and selectivity for use in such separations.
Liquid chromatography (LC) is a technique capable of separating a sample mixture into its components. The sample is transported through a separating column, using a mobile phase carrier, which resolves or separates the sample components such that they elute from the column in seriatim. A detector provides an electrical signal in response to each component, which signal appears graphically as a peak.
Chromatographic resolution of the components or peaks is dependent on many factors. Three primary factors that contribute to resolution (R.sub.s) are relative retention (K'), separation selectivity (.alpha.), and column efficiency (N). The factors are related to resolution by the equation ##EQU1## The mobile phase composition has a dominant influence on retention and selectivity. Changes in the mobile phase strength primarily affect the retention. Changes in the ingredients used in the mobile phase affects the specific chemical interactions in the resultant separation and therefore the selectivity. In the past, mobile phase strength and especially selectivity were adjusted empirically with an open-ended approach.
Chemical data is now available which classifies mobile phase solvents used in reverse phase and bonded phase LC according to the several selectivity interactions; proton acceptor, proton donor and dipole interaction. Similar data is available for the selectivity interactions or localization effects that affect separations in liquid-solid chromatography. These effects include a nonlocalizing solvent, a basic localizing solvent, and a nonbasic localizing solvent. These interactions are summarized in a so-called "selectivity triangle" with each apex representing one of the interactions. Mobile phase solvents are located within the triangle according to the relative contributions of these three interactions to the total solvent strength. Conversely, the location of each solvent is indicative of its selectivity effect.
This "selectivity triangle" has been used as the basis for rational solvent selectivity determinations. Recently, Glajch, Kirkland, Squire and Minor showed, in their article Optimization of Solvent Strength and Selectivity for Reversed-Phase Liquid Chromatography (J. Chromatogr. 199, 57 (1980)), an efficient and systematic technique for optimization of the mobile phase selectivity .alpha. for reversed phase LC separations. This technique uses four solvents, one from each apex of the selectivity triangle and a diluent, in various blends, which exhibit different selectivity but maintain constant retention K', to perform seven experiments. The computerized mapping technique used to facilitate finding the optimum solvent blend for the mobile phase is based on Snee (Experimenting with Mixtures, Chemtech, 9 (Nov.), 702 (1979)).
Even with these prior art techniques, it was difficult and time consuming to select the proper solvent strength (composition) for a particular retention. And since this selection had to be made for each of the three selectivity solvents, it became a slow and inefficient manual operation. In addition, for each of the seven experiments, the total number of sample injections required is one plus the number of components in the sample mixture. This also increased the time required. As a result, most chromatographers did not adopt these techniques and many separations were effected under less than desirable conditions. In those cases where proper conditions were attained, many more experiments than necessary were usually performed to determine such conditions.